http://repub.eur.nl/res/aut/1530/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/30397/ 2008-03-01T00:00:00Z The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced. http://repub.eur.nl/res/pub/2635/ 2002-09-02T00:00:00Z While an important role for the POU domain transcription factor Oct-6 in the developing peripheral nerve has been well established, studies into its exact role in nerve development and regeneration have been hampered by the high mortality rate of newborn Oct-6 mutant animals. In this study we have generated a Schwann cell-specific Oct-6 allele through deletion of the Schwann cell-specific enhancer element (SCE) in the Oct-6 locus. Analysis of mice homozygous for this allele (ΔSCE allele) reveals that rate-limiting levels of Oct-6 in Schwann cells are dependent on the SCE and that this element does not contribute to Oct-6 regulation in other cell types. We demonstrate a Schwann cell autonomous function for Oct-6 during nerve development as well as in regenerating nerve. Additionally, we show that Krox-20, an important regulatory target of Oct-6 in Schwann cells, is activated, with delayed kinetics, through an Oct-6-independent mechanism in these mice. http://repub.eur.nl/res/pub/9965/ 2002-01-01T00:00:00Z While an important role for the POU domain transcription factor Oct-6 in the developing peripheral nerve has been well established, studies into its exact role in nerve development and regeneration have been hampered by the high mortality rate of newborn Oct-6 mutant animals. In this study we have generated a Schwann cell-specific Oct-6 allele through deletion of the Schwann cell-specific enhancer element (SCE) in the Oct-6 locus. Analysis of mice homozygous for this allele (deltaSCE allele) reveals that rate-limiting levels of Oct-6 in Schwann cells are dependent on the SCE and that this element does not contribute to Oct-6 regulation in other cell types. We demonstrate a Schwann cell autonomous function for Oct-6 during nerve development as well as in regenerating nerve. Additionally, we show that Krox-20, an important regulatory target of Oct-6 in Schwann cells, is activated, with delayed kinetics, through an Oct-6-independent mechanism in these mice. http://repub.eur.nl/res/pub/2607/ 2001-07-09T00:00:00Z The transcription factor GATA6 is expressed in the fetal pulmonary epithelium of the developing mouse lung and loss of function studies strongly suggested that it is required for proper branching morphogenesis and epithelial differentiation. We have further investigated the role of GATA6 in this process by utilizing a pulmonary epithelium specific promoter to maintain high levels of GATA6 protein during fetal lung development. Transgenic mice expressing Gata6 cDNA under the control of the human Surfactant Protein-C (SP-C) promoter were generated and their lungs were analyzed during fetal stages. Transgenic lungs exhibit branching defects as early as embryonic day (E) 14.5 and molecular analysis just before birth (E18.5) shows a lack of distal epithelium differentiation whereas proximal epithelium is unaffected. Electron microscopic analysis and glycogen staining confirm the lack of differentiation to mature Type II cells. Thus, elevated levels of GATA6 protein affect early lung development and in analogy to other GATA factors in other tissues, GATA6 also plays a crucial role in the terminal differentiation in this case of the distal pulmonary epithelium. http://repub.eur.nl/res/pub/23390/ 2001-04-25T00:00:00Z Different members of the GAT A family of transcription factors have been studied extensively in our lab. The role of GAT A-I in the differentiation of erythroid blood cells and of GATA-3 during T-lymphocyte development are two typical examples. GATA-6 is the most recently characterized member of the family. Based on its expression pattern during mouse embryonic developmentfor a role for GATA-6 in cardiogenesis had been speculated. To investigate what role GATA-6 may play during embryogenesis we used targeted inactivation of the gene in Embryonic Stem (ES) cells (chapter 2). Unexpectedly, homozygote mutant embryos die just after implantation at embryonic day 5.5. Generation of chimeric embryos in which the GATA-6 mutant cell population was confmed in either the embryo or to the extraembryonic tissues revealed that the primary defect in GATA-6 null embryos lies in an extraembryonic cell lineage. Further in vivo and in vitro analysis of the mutant embryos suggested that the affected lineage is the visceral yolk sac endoderm, a derivative of the primitive endoderm. Cardiogenesis could not be directly studied since mutant embryos die well before heart development starts (embryonic day 8.5). However, in chimeric embryos, GATA-6 -/- ES cells give rise to cardiomyocytes in apparently normal hearts, possibly due to redundant functions with the coexpressed GATA-4 and -5. In contrast, GATA-6 is the only member of the family that is expressed in the lung endoderm. Following on a published observation showing no contribution of GATA-6 null ES cells to the lung epithelium, we decided to generate more highly chimeric embryos to analyze the development of the lung, which is a derivative of another endoderm lineage, the definitive endoderm (chapter 3). Surprisingly, we found that lung endoderm can be formed from GATA-6 mutant cells. However, this mutant endoderm has subsequent morphogenetic and differentiation defects. The importance of GATA-6 protein levels during lung development was confirmed by a different approach. The gene was overexpressed in transgenic mice with a pulmonary epithelium specific promoter (chapter 4). High levels of the protein resulted in branching defects and more interestingly in a block oflung endoderm differentiation to distal alveolar epithelium. http://repub.eur.nl/res/pub/9590/ 2001-01-01T00:00:00Z Recent loss-of-function studies in mice show that the transcription factor GATA6 is important for visceral endoderm differentiation. It is also expressed in early bronchial epithelium and the observation that this tissue does not receive any contribution from Gata6 double mutant embryonic stem (ES) cells in chimeric mice suggests that GATA6 may play a crucial role in lung development. The aim of this study was to determine the role of GATA6 in fetal pulmonary development. We show that Gata6 mRNA is expressed predominantly in the developing pulmonary endoderm and epithelium, but at E15.5 also in the pulmonary mesenchyme. Blocking or depleting GATA6 function results in diminished branching morphogenesis both in vitro and in vivo. TTF1 expression is unaltered in chimeric lungs whereas SPC and CC10 expression are attenuated in abnormally branched areas of chimeric lungs. Chimeras generated in a ROSA26 background show that endodermal cells in these abnormally branched areas are derived from Gata6 mutant ES cells, implicating that the defect is intrinsic to the endoderm. Taken together, these data demonstrate that GATA6 is not essential for endoderm specification, but is required for normal branching morphogenesis and late epithelial cell differentiation. http://repub.eur.nl/res/pub/9125/ 1999-01-01T00:00:00Z The gene coding for the murine transcription factor GATA6 was inactivated by insertion of a beta-galactosidase marker gene. The analysis of heterozygote GATA6/lacZ mice shows two inductions of GATA6 expression early in development. It is first expressed at the blastocyst stage in part of the inner mass and in the trophectoderm. The second wave of expression is in parietal endoderm (Reichert's membrane) and the mesoderm and endoderm that form the heart and gut. Inactivation leads to a lethality shortly after implantation (5.5 days postcoitum). Chimeric experiments show this to be caused by an indirect effect on the epiblast due to a defect in an extraembryonic tissue.